The incidence of Human papillomavirus (HPV) infections is high in the developing countries. In India, more than 134,000 new cases and nearly 74,000 deaths due to cervical cancer are reported every year according to the report of the WHO/ICO HPV Information Centre, June 2010.
In India, cervical and breast cancer are most frequent among the cancer cases in women particularly in the age group of 15-44 years. About 7.9% of women in the general population harbor HPV infection and most incidences of invasive cervical cancers are caused by HPV16 and HPV18. Mixed infections with other HPV genotypes are common Candidate vaccines that confer broad protective efficacy against multiple genotypes is the way forward for effective HPV vaccination. The HPV vaccines using the L1 capsid proteins are type specific for HPV16, HPV18, HPV6 and HPV11 and fail to offer complete protection against infection caused by other genotypes such as HPV31, HPV35, HPV52, HPV58 etc. which are also associated with progression of HPV infection to cervical carcinoma. There are no HPV vaccines available commercially as yet, that offers cross protection against infection by multiple HPV genotypes. Two preventive vaccines comprising recombinant HPV L1 virus-like particles (VLPs) have been licensed. They target only two of the approximately 15 known oncogenic HPV types. Nearly 70% of cervical cancer cases are attributed to HPV16 and HPV18, and there is evidence for some degree of cross-protection against the other closely related genotypes of HPV. Other approaches to the development of broadly protective HPV vaccines include multivalent vaccines using L1 capsid protein of at least nine HPV oncogenic viruses, with no added cost advantage and hence the cost of these vaccines would preclude sustained global delivery particularly in third world countries where the incidence of HPV infection is high. Other approaches have used the HPV L1 antigens in combination with HPV L2 capsid protein which have broadly neutralizing epitopes. However, the L2 capsid protein is by itself poorly immunogenic necessitating alternate strategies of combining the epitopes derived from L2 protein in combination with HPV L1 capsid protein, or in combination with other viral antigens that have the ability to assemble into Virus-Like Particles (VLPs). The rationale for design of such chimeras is for increasing the epitope density of the heterologous peptides on the backbone of the VLPs thereby increasing their immunogenicity. Chimeric constructs of HPV16 L2 epitopes with the HbsAg (Hepatitis B surface antigen) to assemble into virus like particles (VLPs) has been described in PCT/IN2009/000333.
Hepatitis E virus (HEV) is an enterically transmitted virus that is responsible for endemic hepatitis as well as sporadic acute hepatitis. HEV-infected persons exhibit a wide clinical spectrum ranging from asymptomatic infection to fulminant hepatitis. The clinical features of hepatitis E virus with icterus, high fever, hepatomegaly, and pruritus are similar to those of acute viral hepatitis caused by other hepatotropic viruses. The viral infection is commonly diagnosed by laboratory findings of elevated serum bilirubin, increase in liver enzymes and mild increases in alkaline phosphatase activity. The incidence of HEV is high in the developing countries (Emerson and Purcell, 2007; Chandra et al., 2008). HEV is an important aetiological agent for sporadic fulminant hepatic failure (FHF) in developing countries [Nanda et al., 1994]. The HEV infection accounts for more than 50% of acute viral hepatitis cases with a case fatality of 0.2% to 4% in the general population and upto 20% in pregnant women especially during the third trimester of pregnancy (Guu et al., 2009). High mortality, particularly during the third trimester of pregnancy, following fulminant hepatic failure, is characteristic of HEV infection in pregnant women (Khuroo et al., 1981) and may be related to hormonal changes in pregnancy (Lindemann et al., 2010). In studies involving pregnant women, it was shown that HEV accounted for nearly 37% of acute viral hepatitis infection and 81% of cases of fulminant hepatitis (Beniwal et al., 2003). An obstetric complication, such as premature rupture of membranes and intrauterine growth restriction is common during HEV infection is pregnancy [Kumar et al., 2004]. In HEV infection during third trimester of pregnancy, death is usually due to fulminant hepatitis or obstetric complications (Tsega et al., 1993). Vertical transmission of the virus with consequent morbidity and mortality of infants is also common with third trimester hepatitis E infections (Khuroo et al., 1995). The reasons for high mortality in pregnant women have not been understood clearly. HEV infection may also be associated with severe disease in persons with preexisting liver disease [Kumar Acharya et al., 2007]. Chronic infection with HEV is rare, but is more frequent in HIV-infected persons [Dalton et al., 2009]. Hepatitis outbreaks in developing countries have been caused primarily by HEV genotype 1. Epidemic outbreaks in Mexico and Western Africa were caused by genotype 2. Sporadic cases in Asia were caused by genotype 4. So far only genotypes 1 and 2 have occurred in humans, whereas genotypes 3 and 4 have also been isolated in animals. HEV has only one serotype (Mushahwar, 2008) which means that a candidate vaccine of one genotype can cross protect infection caused by other genotypes of the HEV. The use of Hepatitis E virus antigens including the ORF2, either the full length or truncated protein is known in the prior art (Amini-Bavil-Olyaee et al., 2009). The use of HEV viral antigens as vaccine for prophylaxis of HEV infection has been disclosed. A recombinant vaccine of HEV ORF2 that has been produced in E. coli has also been found to be safe for administration in humans (Zhu et al., 2010). A combination of the Hepatitis E and HPV vaccines for comprehensive healthcare of women is not disclosed in the prior art. Hence it was required to be demonstrate experimentally the feasibility of developing a combination vaccine where there is no antigenic interference when the hepatitis virus and HPV antigens are administered concomitantly either as an admixture that is mixed extemporaneously or as a chimeric antigen that carries both the vaccine epitopes in a single polypeptide expressed as a virus like particle (VLP). The development and use of such combinations is not known in the prior art.
The common problem that is encountered in the development of chimeric vaccines with the other viral antigens is the limitation of the scaffold antigen to accommodate heterologous protein sequences without affecting the native structural conformation that is required for immunogenicity of both the vaccines, especially if it were to be assembled as a virus like particle. Apart from the scaffold antigen, the sequence and the structural conformation of the heterologous protein for preparing the chimeric antigen is important. Also critically important is the location on the scaffold antigen where the heterologous sequence is to be added. Hence these factors cannot be pre-determined until the chimeric constructs are made and tested. To put it simply, what works for a pair of antigens would not be identical to another pair of antigens and hence this information cannot be extrapolated from the prior art describing similar design of chimeric antigens. The full length ORF2 protein of Hepatitis E consists of 660 amino acids. The neutralizing epitopes of the virus are not present largely at the N-terminus and C-terminus of the protein. Large amino acid deletions at the N- and the C-terminus do not diminish the ability of the protein to assemble into VLPs. Rational design of HPV-HEV chimeras involve the insertion of the heterologous HPV L2 broadly neutralizing epitopes in any region of the HEV ORF2 protein that does not affect the native structure of the protein and without compromising the immunogenicity of both the viral antigens. The insertion of the HPV L2 epitopes of desired sequences into any region of the HEV ORF2 protein that does not diminish the ability of the HEV antigen to assemble into VLPs, and at the same time display the heterologous HPV L2 epitopes on the surface of the chimeric VLP would be an ideal choice for vaccines against both HPV and HEV. A combination of HEV antigens with HEV-HPVL2 chimeric antigen and/or with the HPV16 and HPV18 L1 capsid antigens as an admixture of the two antigens is also a very effective vaccine combination. Addition of HbsAg or chimeric HbsAg-HPVL2 whose method of production is described in PCT/IN2009/000333 to the HEV and/or HEV-L2 antigens is also not known in the prior art. ORF3 of HEV which is also an immunogenic structural protein is a good vaccine candidate that can be included as an antigen in such a vaccine combination. The addition of Tdap or DTap (diphtheria toxoid, tetanus toxoid and acellular pertussis antigens) or DTwP, wherein the acellular pertussis antigens are replaced by whole cell pertussis antigens to the aforementioned vaccine antigens is also novel and is not described in the prior art. The discovery and development of such vaccine combination is also very significant for the reasons mentioned below.
The mortality rate due to neonatal tetanus is high globally and especially so in the developing countries. It is easily preventable as antibodies against tetanus toxin are actively transported by the placenta from the immunized mother to her fetus. This provides passive protection against tetanus during the neonatal period and for a few months following birth. The mortality rate due to neonatal tetanus can be reduced with immunization of pregnant women or in women of childbearing age. It has been demonstrated that the use of two or more doses of tetanus toxoid during pregnancy could prevent neonatal tetanus (Schofield et al., 1961) Tetanus toxoid vaccination of pregnant women has been included in the WHO's Expanded Program on Immunization. Similarly, vaccination with Tdap (tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis) vaccines is also recommended for pregnant women. It is recommended that one dose of Tdap be administered during the third trimester or late in the second trimester (after 20 weeks gestation). If not administered during pregnancy, Tdap should be administered immediately postpartum. When Tdap is administered during pregnancy, at time of delivery the mother will be protected, making her less likely to transmit pertussis to her infant, and transplacental maternal antibodies will likely protect the infant against pertussis in early life. The preferred schedule in pregnant women is two doses of Td separated by 4 weeks, and a dose of Tdap 6 months after the second dose (post-partum). It is recommended that adolescents and/or women of active child bearing age should receive a dose of Tdap preferably before pregnancy. Whole cell pertussis vaccine can also be administered in lieu of acellular pertussis vaccine. Hence Tdap and/or tetanus toxoid vaccines are a good option to be included in the combination vaccine for women along with HPV and HEV antigens.
Such a combination vaccine for Hepatitis E with tetanus toxoid or with Tdap with and without HPV antigens is novel and is not commercially available or it is disclosed in any prior art. In the current invention, methods are disclosed on the development of a combination vaccine of HEV and HPV. The rationale for combining the aforementioned antigens either in a single formulation as an admixture, or administered concomitantly is to improve the overall health outcome in women, as cervical cancer ranks high as the most frequent cause of cancer among women in India, and also for the fact that hepatitis E infection causes high mortality in pregnant women particularly in the third trimester of pregnancy. The HPV-HEV vaccine combination can be expected to decrease the mortality and morbidity due to HPV and HEV infections and to improve the overall health in this segment of the population and as recommended, vaccination with Tdap to pregnant women reduces infant mortality due to tetanus and pertussis. Such combination vaccines are not only useful to provide protection against multiple pathogens, but are very desirable as they reduce the number of immunizations required. Combination vaccines also help to lower the manufacturing and distribution cost, making the vaccines cost-effective and affordable. Affordability of vaccines increases acceptance and coverage rates. The use of suitable adjuvants in the vaccine formulations also reduces the amount of antigen required and helps in the manufacture of low-cost vaccines thus conferring a distinct economic advantage. No antigenic interference has been observed with the co-administration of all the aforementioned antigens and hence combination of different antigens can be made. For the production of recombinant antigens, Pichia pastoris as recombinant expression host is advantageous at industrial scale as it is cost effective for large scale manufacture compared to other eukaryotic expression systems. Recombinant proteins derived from Pichia pastoris have been successfully commercialized and have been found safe for human use. Use of this expression system for the manufacture of both the HPV and HEV antigens is therefore safe and highly cost-effective for production of vaccines for HPV and HEV.